1. Field of the Invention
This invention is in the field of medicinal chemistry. In particular, the invention relates to dipeptides which are potent inhibitors of apoptosis. The invention also relates to the use of these dipeptides for reducing or treating apoptotic cell death.
2. Description of Background Art
Organisms eliminate unwanted cells by a process variously known as regulated cell death, programmed cell death or apoptosis. Such cell death occurs as a normal aspect of animal development as well as in tissue homeostasis and aging (Glucksmann, A., Biol. Rev. Cambridge Philos. Soc. 26:59-86 (1951); Glucksmann, A., Archives de Biologie 76:419-437 (1965); Ellis et al., Dev. 112:591-603 (1991); Vaux et al., Cell 76:777-779 (1994)). Apoptosis regulates cell number, facilitates morphogenesis, removes harmful or otherwise abnormal cells and eliminates cells that have already performed their function. Additionally, apoptosis occurs in response to various physiological stresses, such as hypoxia or ischemia (PCT published application WO96/20721).
There are a number of morphological changes shared by cells experiencing regulated cell death, including plasma and nuclear membrane blebbing, cell shrinkage (condensation of nucleoplasm and cytoplasm), organelle relocalization and compaction, chromatin condensation and production of apoptotic bodies (membrane enclosed particles containing intracellular material) (Orrenius, S., J. Internal Medicine 237:529-536 (1995)).
Apoptosis is achieved through an endogenous mechanism of cellular suicide (Wyllie. A. H., in Cell Death in Biology and Pathology, Bowen and Lockshin, eds., Chapman and Hall (1981), pp. 9-34). A cell activates its internally encoded suicide program as a result of either internal or external signals. The suicide program is executed through the activation of a carefully regulated genetic program (Wylie et al., Int. Rev. Cyt. 68: 251 (1980); Ellis et al., Ann. Rev. Cell Bio. 7: 663 (1991)). Apoptotic cells and bodies are usually recognized and cleared by neighboring cells or macrophages before lysis. Because of this clearance mechanism, inflammation is not induced despite the clearance of great numbers of cells (Orrenius, S., J. Internal Medicine 237:529-536 (1995)).
Mammalian interleukin-1xcex2 (IL-1xcex2) plays an important role in various pathologic processes, including chronic and acute inflammation and autoimmune diseases (Oppenheim, J. H. et. al. Immunology Today, 7, 45-56 (1986)). IL-1xcex2 is synthesized as a cell associated precursor polypeptide (pro-IL-1xcex2) that is unable to bind IL-1 receptors and is biologically inactive (Mosley et al., J. Biol. Chem. 262:2941-2944 (1987)). By inhibiting conversion of precursor IL-1xcex2 to mature IL-1xcex2, the activity of interleukin-1 can be inhibited. Interleukin-1xcex2 converting enzyme (ICE) is a protease responsible for the activation of interleukin-1xcex2 (IL-1xcex2) (Thornberry, N. A., et al., Nature 356: 768 (1992); Yuan, J., et al., Cell 75: 641 (1993)). ICE is a substrate-specific cysteine protease that cleaves the inactive prointerleukin-1 to produce the mature IL-1. The genes that encode for ICE and CPP32 are members of the mammalian ICE/Ced-3 family of genes which presently includes at least twelve members: ICE, CPP32/Yama/Apopain, mICE2, ICE4, ICH1, TX/ICH-2, MCH2, MCH3, MCH4, FLICE/MACH/MCH5, ICE-LAP6 and ICErelIII. The proteolytic activity of this family of cysteine proteases, whose active site cysteine residue is essential for ICE-mediated apoptosis, appears critical in mediating cell death (Miura et al., Cell 75: 653-660 (1993)). This gene family has recently been named caspases (Alnernri, E. S. et al. Cell, 87:171 (1996)).
IL-1 is also a cytokine involved in mediating a wide range of biological responses including inflammation, septic shock, wound healing, hematopoiesis and growth of certain leukemias (Dinarcllo, C. A., Blood 77:1627-1652(1991); diGiovine et al., Immunology Today 11:13 (1990)).
Many potent caspase inhibitors have been prepared based on the peptide substrate structures of caspases. However, in contrast to their potency in vitro, no inhibitors with good efficacy (IC50 less than 1 xcexcM) in whole-cell models of apoptosis have been reported (Thomberry, N. A. Chem. Biol. 5:R97-103 (1998)). Therefore the need exists for cell death inhibitors that show efficacy (IC50 less than 1 xcexcM) in whole-cell models of apoptosis and are active in animal models of apoptosis. These inhibitors thus can be employed as therapeutic agents to treat disease states in which regulated cell death and the cytokine activity of IL-1 play a role.
WO 93/05071 disclosed peptide ICE inhibitors with the formula:
Zxe2x80x94Q2xe2x80x94Aspxe2x80x94Q1
wherein Z is an N-terminal protecting group; Q2 is 0 to 4 amino acids such that the sequence Q2-Asp corresponds to at least a portion of the sequence Ala-Tyr-Val-His-Asp; Q1 comprises an electronegative leaving group. Exemplary dipeptides are Boc-His-Asp-CH2F, Boc-Tyr-Asp-CH2F, Boc-Phe-Asp-CH2F, Ac-His-Asp-CH2F, Ac-Tyr-Asp-CH2F, Ac-Phe-Asp-CH2F, Cbz-His-Asp-CH2F, Cbz-Tyr-Asp-CH2F and Cbz-Phe-Asp-CH2F.
WO 96/03982 disclosed aspartic acid analogs as ICE inhibitors with the formula: 
wherein R2 is H or alkyl; R3 is a leaving group such as halogen; R1 is heteroaryl-CO or an amino acid residue.
U.S. Pat. No. 5,585,357 disclosed peptidic ketones as ICE inhibitors with the formula: 
wherein n is 0-2; each AA is independently L-valine or L-alanine; R1 is selected from the group consisting of N-benzyloxycarbonyl and other groups; R8, R9, R10 are each independently hydrogen, low alkyl and other groups.
Revesz et al. (Tetrahedron Lett. 35, 9693-9696, 1994) reported the preparation of ethyl ester tripeptide: 
as a prodrug of the corresponding acid which is a potent ICE inhibitor.
The invention relates to dipeptides of formula I: 
wherein R1 is an N-terminal protecting group; AA is a residue of any natural or non-natural xcex1-amino acid, xcex2-amino acid, derivatives of an xcex1-amino acid or xcex2-amino acid; R2 is H or CH2R4 where R4 is an electronegative leaving group, and R3 is alkyl or H, provided that AA is not His, Tyr, Pro or Phe.
The invention relates to the discovery that the dipeptide-based caspase inhibitors represented by Formula I, though less potent in enzyme assays than tri- and tetrapeptide inhibitors on the enzymes, are surprisingly potent inhibitors of apoptosis in cell based systems. These compounds are systemically active in vivo and are potent inhibitors of antiFas-induced lethality in a mouse liver apoptosis model and have robust neuroprotective effects in a rat model of ischemic stroke.
The invention also relates to the use of the dipeptides of the invention for reducing, preventing or treating maladies in which apoptotic cell death is either a causative factor or a result. Examples of uses for the present invention include protecting the nervous system following focal ischemia and global ischemia; treating neurodegenerative disorders such as Alzheimer""s disease, Huntington""s Disease, Orion diseases, Parkinson""s Disease, multiple sclerosis, amyotrophic lateral sclerosis, ataxia, telangiectasia, and spinobulbar atrophy; treating heart disease including myocardial infarction, congestive heart failure and cardiomyopathy; treating retinal disorders; treating autoimmune disorders including lupus erythematosus, rheumatoid arthritis, type I diabetes, Sjogren""s syndrome and glomerulonephritis; treating polycystic kidney disease and anemia/erythropoiesis; treating immune system disorders, including AIDS and SCIDS; reducing or preventing cell, tissue and organ damage during transplantation; reducing or preventing cell line death in industrial biotechnology; reducing or preventing alopecia (hair loss); and reducing the premature death of skin cells.
The present invention provides pharmaceutical compositions comprising a compound of Formula I in an effective amount to reduce apoptotic cell death in an animal.
The present invention also provides preservation or storage solutions for manmnralian organs or tissue, or growth media for mammalian or yeast cells, wherein an effective amount of a compound of Formula I is included in said solutions or media in order to reduce apoptotic cell death in said organs, tissue or cells.